Deliverables Distribution of all sst subtypes With respect to the receptor subtype selective antibodies, the consortium has generated in rabbits and characterized polyclonal antibodies for rodent and human sst1, sst2A, sst2B, sst3, sst4 and sst5 antisera. With these antibodies we have established the expression pattern in more than 200 distinct areas of the rat brain for the sst subtypes. They all showed a broad overlapping expression pattern. Only few structures were almost free of sst immunstaining, such as the Edinger-Westphal nucleus or layer I of the cerebral cortex. In general all subtypes but sst5, which we could not reliably detect in adult brain, were prominantly present in olfactory structures, suggesting roles in processing chemical sensory information. Moreover, sst1 was abundantly seen in the deep layers of cerebral cortex and granule cell layer of the cerebellum, while sst2A and sstB are probably the most ubiquitously expressed variants. At the cellular level sst subtypes were present on neurons with little evidence At the subcellular level we found sst1 frequently associated with neuronal fibers and terminals, sst2A, sstB, and sst4 with somata and dendrites and sst3 with neuronal cilia. Sites of sst1, sst2A, sst2B and sst4 localization were usually seen to be closely opposed by somatostatin-containing terminals. These observation indicates that the somatostatin signalling system fulfils the anatomical requirements for being functional and modulating a variety of transmission processes. Map of sst subtype co-localization with neurotransmitters The interaction of somatostatin with other neurotransmitters may reveal therapeutic targets. To further extent our previous observation of the spatial relationship of the somatostatin signalling system and the biogenic amines we now investigated the co-localization of somatostatin receptors and tryptophane hydroxylase (TPH), a marker for serotonergic neurotransmission. The cell bodies of serotonergic neurons are located in the brain stem restricted to 9 discrete cell clusters (B1-B9) within the raphe nuclei and adjacent nuclear groups. Therefore, we examined these brain regions using an immunohistochemical double labelling method to detect cells, co-expressing sst subtypes and TPH. Our results demonstrate that TPH positive cells have the highest degree of co-localization with sst2B immunoreactivity in neurons of the cell groups B1-B9. Still about 46% of sst1 and TPH-positive cells co-localize in the cell groups B1-B9 and nearly the same level of co-localization was detected for sst4 and TPH-positive cells in the cell groups B1-B3 and B9. Lower numbers of sst4 and TPH co-expressing cells were detected in cell clusters B5+B8, B6+B7, B7_CG and B4. A low degree of co-localization was also detected for sst2A and TPH expressing cells located in brain regions B3, B9, B2, B1 and B5+B8. Very low numbers of co-expressing cells were detected in the cell clusters B6+B7. In region B7_CG no sst2A and TPH co-expressing cells were observed. A minimal co-localization of sst3 and TPH immunoreactivity was detected in the cell clusters B3 and B7_CG. No double labelled cells were found in the regions B4, B5+B8, B6+B7 and in the cell groups B1_RPa, B1_CVL, B2 and B9 even no sst3 positive neuronal cilia were observed. Our data suggest that interactions between the somatostatinergic and the serotonergic systems mainly involve the receptor subtypes sst2B, sst1, sst4 and to a lower extend sst2A, whereas sst3 seems not to play an important role in this interaction. Another site where we studied functional interaction of somatostatin with other signalling systems was the rat hypothalamus, a major site regulating feeding behaviour. During the last reporting period we have shown that in many hypothalamic regions somatostatin receptor subtypes are expressed in leptin-responsive neurons. We now have addressed the functional importance of this co-localization by icv administration of leptin and somatostatin to experimental rats in comparison to control animals. By western blotting experiments using hypothalamic protein extracts we demonstrated that somatostatin and sst1, sst2 or sst3 selective agonists inhibited leptin mediated phosphorylation of the signalling molecule STAT3, while an sst4 selective agonist was ineffective. We also examined the effect of somatostatin agonists histochemically on the leptin-induced nuclear translocation of STAT3 in hypothalamic nuclei known to participate in feeding regulation. Somatostatin inhibited STAT3 translocation significantly in the ventromedial hypothalamic nucleus (VMH), the lateral hypothalamic area (LHA) and the dorsomedial hypothalamic nuclus (DMH) to various extents. It did not significantly so in the arcuate nucleus (ARC). These effects were also seen with sst3 agonists and could be attenuated by an sst3 antagonists. Sst1 and sst2 agonists showed still significant, but weaker effects in the aforementioned nuclei. In addition they could also block STAT3 in the ARC. As a behavioural correlate we also measured inhibition of leptins anorectic effect by somatostatin agonists. It turned out that somatostatin itself and sst1, sst2 and sst3 agonists were effective while sst4 agonists did not elicit an effect. These data suggest that somatostatin is a modulator of leptins anorectic action. Role of sst2 as a tumour suppresser The purpose of the present study was to analyse whether somatostatin was able to inhibit the intracrine-induced growth of rat pancreatic cancer cells AR4-2J expressing the HMW FGF-2 of 210 aa. This cell line is characterized by a high expression of endogenous sst2.The FGF-2 isoform of 210 amino acids (HMW FGF-2) contains a nuclear localization sequence (NLS) and is targeted to the nucleus. This FGF-2 isoform allows cells to growth in low serum concentrations through still unknown mechanisms called intracrine regulations. The existence of molecules acting as negative regulators of the intracrine-induced cell growth has not been explored. We demonstrated that in AR4-2J cells stably expressing HMW FGF-2, activation of the somatostatin receptor subtype sst2 by the somatostatin analogue RC-160 in serum-deprived medium inhibits the mitogenic effect of the HMW FGF-2, without affecting growth of control cells. The signaling pathway implicates GÑi/JAK2/SHP-1. The GÑi inhibitor pertussis toxin and the JAK2 inhibitor AG490 abrogate the inhibitory effect of RC-160 on HMW FGF-2-induced cell growth. Co-mmunoprecipitation studies demonstrate the constitutive association of JAK2 and SHP-1, and RC-160 induces a rapid activation of both proteins followed by the dissociation of the complex. AG490 prevents the RC-160 induced SHP-1 activation indicating the implication of JAK2 in this process. JAK2 and SHP-1 are immunoprecipitated with sst2 in basal conditions indicating the existence of a functional signaling complex at the receptor level. In summary, these data provide the first evidence that: - The intracrine-induced proliferation can be reversed by extracellular acting polypeptides such as somatostatin. - Sst2 inhibits HMW FGF growth signaling by activating the JAK2/SHP-1 pathway in AR-42J cells. We have showed that in vivo sst2 gene transfer results in a significant antitumor effect characterized by both an increase of apoptosis, and an inhibition of cell proliferation. The present study was conducted to identify antitumoral bystander mechanisms elicited by sst2 expression after in vivo sst2 gene transfer in nude mice bearing pancreatic carcinoma. Functional map of sst subtypes in the brain Hypothalamic leptinoceptive neurons can be visualized by histochemical demonstration of leptin-induced nuclear translocation of the signaling molecule STAT3. We investigated the relation of the leptinoceptive neurons to the somatostatin signaling system. With double-labeling immunohistochemistry we have studied the co-localization of leptin-activated transcription factor STAT3 with somatostatin receptor subtypes sst1, sst2A, sst2B, sst3 and sst4 or the neuropeptide itself in rat hypothalamus. Immunoreactivity for all the entities was widely distributed throughout the entire hypothalamus. Despite the wide distribution, only few cases of co-localization of somatostatin with leptin-activated STAT3 were detected in the paraventricular, arcuate and dorsomedial nuclei. A moderate to high degree of co-localization of nuclear STAT3 and all investigated subtypes of somatostatin receptors was found in the lateral and dorsal hypothalamic areas and in the dorsomedial hypothalamic nucleus. Immunoreactivity for sst1, sst2B and sst4 was present in STAT3-containing nuclei of the paraventricular, periventricular, arcuate and ventromedial hypothalamic neurons as well as in the retrochiasmatic and posterior hypothalamic areas. Despite the wide distribution of sst2A in rat hypothalamus, few events of co-localization with leptin-activated STAT3 were observed in the dorsomedial nucleus and in the lateral and dorsal hypothalamic areas only. Many leptin-responsive neurons of the dorsal, lateral, periarcuate, perifornical and posterior hypothalamic areas as well as in the ventromedial and dorsomedial hypothalamic nuclei displayed sst3 immunoreactivity at their neuronal cilia. These results provide a strong anatomical evidence for the direct interaction of leptin and the somatostatinergic system in neuroendocrine control loops such as the energy homeostasis, growth or stress response. By detecting nuclear STAT3 translocation in rat hypothalamus we investigated the possible influence of somatostatin on leptin central action. We demonstrated that somatostatin as well as sst1, sst2 and sst3 selective agonists, administered centrally prior to leptin injection, inhibited the activation of STAT3 that is resulted in the decrease in number of STAT3 positive cells and decrease in the amount of phosphorylated STAT3 in rat hypothalamus. Furthermore, we showed that somatostatin inhibits the activation of STAT3 by leptin in dose-dependent manner. Additionally, somatostatin and sst1, sst2 and sst3 agonists counteract the suppression of 24-hour food intake caused by leptin. In contrast, sst4 selective agonist, applied before leptin injection did not cause any change in STAT3 activation as well as in anorexia induced by leptin. Searching for OpenAIRE data... There was an error trying to search data from OpenAIRE No results available
